Importance of a serine proximal to the C(4a) and N(5) flavin atoms for hydride transfer in choline oxidase

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer.     

Mechanistic studies of the SufS-SufE cysteine desulfurase: evidence for sulfur transfer from SufS to SufE

SufS is a cysteine desulfurase of the suf operon shown to be involved in iron-sulfur cluster biosynthesis under iron limitation and oxidative stress conditions. The enzyme catalyzes the conversion of L-cysteine to L-alanine and sulfide through the intermediate formation of a protein-bound cysteine persulfide in the active site. SufE, another component of the suf operon, has been previously shown to bind tightly to SufS and to drastically stimulate its cysteine desulfurase activity. Working with Escherichia coli proteins, we here demonstrate that a conserved cysteine residue in SufE at position 51 is essential for the SufS/SufE cysteine desulfurase activity. Mass spectrometry has been used to demonstrate (i). the ability of SufE to bind sulfur atoms on its cysteine 51 and (ii). the direct transfer of the sulfur atom from the cysteine persulfide of SufS to SufE. A reaction mechanism is proposed for this novel two-component cysteine desulfurase.     

Mechanistic studies on the flavin:NADH reductase (PrnF) from Pseudomonas fluorescens involved in arylamine oxygenation

We report the mechanistic studies of a FAD:NADH reductase (PrnF) involved in arylamine oxygenation. PrnF catalyzes the reduction of FAD via a sequential ordered bi-bi mechanism with NADH as the first substrate to bind and FADH(2) as the first product to be released. The residues Asp145 and His146 are proposed as catalytic acid/base residues for PrnF based on pH profile and molecular dynamics simulation studies. These studies provide the first detailed account of the mechanism of the flavin reductase involved in arylamine oxygenation.     

Potential of mean force calculation for the proton and hydride transfer reactions catalyzed by medium-chain acyl-CoA dehydrogenase: effect of mutations on enzyme catalysis

Potential of mean force calculations have been performed on the wild-type medium-chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of the active site of the enzyme reveal that an arginine residue (Arg256), conserved in the substrate-binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme active. This active conformation was used in subsequent computations of the enzymatic reactions. It is known that the catalytic alpha,beta-dehydrogenation of fatty acyl-CoAs consists of two C-H bond dissociation processes: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in the wild-type MCAD demonstrate that the reaction proceeds by a stepwise mechanism with a transient species. The activation barriers of the two steps differ by only approximately 2 kcal/mol, indicating that both may contribute to the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barriers can be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along the reaction path reveals that Arg256 plays a key role in maintaining the reaction center hydrogen-bonding network involving the thioester carbonyl group, which stabilizes transition states as well as the intervening transient species. Mutation of this arginine residue to glutamine increases the activation barrier of the hydride transfer reaction by approximately 5 kcal/mol, and the present simulations predict a substantial loss of catalytic activity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioester moiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. In contrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relative orientation of the substrate and the cofactor in the active site; as a result, this mutation has very little effect on the overall reaction barrier, and this is consistent with the experimental data. This study demonstrates that significant insights into the catalytic mechanism can be obtained from simulation studies, and the results can be used to design novel mechanistic probes for the enzyme.     

Mechanistic studies of mouse polyamine oxidase with N1,N12-bisethylspermine as a substrate

In mammalian cells, the flavoprotein polyamine oxidase catalyzes a key step in the catabolism of polyamines, the oxidation of N1-acetylspermine and N1-acetylspermidine to spermidine and putrescine, respectively. The mechanism of the mouse enzyme has been studied with N1,N12-bisethylspermine (BESPM) as a substrate. At pH 10, the pH optimum, the limiting rate of reduction of the flavin in the absence of oxygen is comparable to the k(cat) value for turnover, establishing reduction as rate-limiting. Oxidation of the reduced enzyme is a simple second-order reaction. No intermediates are seen in the reductive or oxidative half-reactions. The k(cat) value decreases below a pK(a) of 9.0. The k(cat)/K(m) value for BESPM exhibits a bell-shaped pH profile, with pK(a) values of 9.8 and 10.8. These pK(a) values are assigned to the substrate nitrogens. The rate constant for the reaction of the reduced enzyme with oxygen is not affected by a pH between 7.5 and 10. Active site residue Tyr430 is conserved in the homologous protein monoamine oxidase. Mutation of this residue to phenylalanine results in a 6-fold decrease in the k(cat) value and the k(cat)/K(m) value for oxygen due to a comparable decrease in the rate constant for flavin reduction. This moderate change is not consistent with this residue forming a tyrosyl radical during catalysis.     

Redesigning the active-site of an acyl-CoA dehydrogenase: new evidence supporting a one-base mechanism

The acyl-CoA dehydrogenases are a family of related enzymes that share high structural homology and a common catalytic mechanism which involves abstraction of an -proton from the substrate by an active site glutamate residue. Several lines of investigation have shown that the position of the catalytic glutamate is conserved in most of these dehydrogenases (the E₂ site), but is in a different location in two other family members (the E₁ site). Using site specific in vitro mutagenesis, a double mutant rat short chain acyl-CoA dehydrogenase (rSCAD) has been constructed in which the catalytic glutamate is moved from the E₂ to the E₁ site (Glu368Gly/Gly247Glu). This mutant enzyme is catalytically active, but utilizes substrate less efficiently than the native enzyme (K_m = 0.6 and 2.0 μM, and V_max = 2.8 and 0.3 s⁻¹ for native and mutant enzyme respectively). In this study we show that both the wild-type and mutant rSCADs display identical stereochemical preference for catalysis—abstraction of the -H_R from the substrate followed by transfer of the β-H_R to the FAD coenzyme. These results, in conjunction with molecular modeling of the native and double mutant SCAD indicate that the catalytic base in the E₁ and E₂ sites are topologically similar and catalytically competent. However, analysis of the ¹H NMR spectra of the incubation products of these two enzymes revealed that, in contrast to the wild-type rSCAD, the Gly368Glu/Gly247Glu rSCAD could not perform γ-proton exchange of the product with the solvent, a property inherent to most acyl-CoA dehydrogenases. It is evident that the base in the mutant enzyme has access to the -H_R but is far removed from the γ-Hs. These findings provide further support for a one base mechanism of - and γ-reprotonation/deprotonation catalysis by acyl-CoA dehydrogenases.     

Identification and properties of 8alpha-(N(1)-histidyl)-riboflavin: the flavin component of thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase.

In addition to 8α-[N(3)-histidyl]-riboflavin which had previously been characterized as the product on condensation of N_α-blocked histidine with 8α-bromotetraacetyl-riboflavin (after removal of the blocking groups), a second histidylflavin isomer is obtained in 20–25% yield of the total histidylflavin fraction. This isomer is identified as 8α-[N(1)-histidyl]-riboflavin by chemical degradation of the histidylflavin analog after alkylation of the imidazole with methyliodide. Acid hydrolysis at high temperature yields 3-methylhistidine, identified by its mobility on high voltage electrophoresis, while Zn reduction yields riboflavin, identified by thin layer chromatography. The properties of synthetic 8α-[N(1)-histidyl]-riboflavin are identical with the histidylriboflavin obtained from thiamine dehydrogenase and β-cyclopiazonate oxidocyclase in pK_a of fluorescence quenching, electrophoretic mobility, and in reduction by sodium borohydride. Thus, both the N(1) and the N(3) histidylriboflavin isomers occur in nature. The compound obtained on acid treatment of 8α-[N(3)-histidyl]-riboflavin (previously thought to be 8α-[N(1)-histidyl]-riboflavin) is shown to differ from the parent compound only in the ribityl side chain.     

A rare disease-associated mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene changes a conserved arginine, previously shown to be functionally essential in short-chain acyl-CoA dehydrogenase (SCAD)

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a serious and potentially fatal inherited defect in the β-oxidation of fatty acids. Approximately 80% of patients with MCAD deficiency are homozygous for a single disease-causing mutation (G985). The remaining patients (except for a few cases worldwide) are compound heterozygous with G985 in one allele. By sequencing of cloned PCR-amplified MCAD cDNA from a G985 compound heterozygous patient, we identified a C-to-T transition at position 157 as the only change in the entire coding sequence of the non-G985 allele. The presence of the T157 mutation was verified in genomic DNA from the patient and her mother by a PCR-based assay. The mutation changes a conserved arginine at position 28 (R28C) of the mature MCAD protein. The effect of the T157 mutation on MCAD protein was investigated by expression of mutant MCAD cDNA in COS-7 cells. On the basis of knowledge about the three-dimensional structure of the MCAD protein, we suggest that the mutation destroys a salt bridge between arginine²⁸ and glutamate⁸⁶, thereby affecting the formation of enzymatically active protein. Twenty-two additional families with compound heterozygous patients were tested in the PCR-based assay. The T157 mutation was identified in one of these families, which had an MCAD-deficient child who died unexpectedly in infancy. Our results indicate that the mutation is rare. It is, however, noteworthy that a homologous mutation has previously been identified in the short-chain acyl-CoA dehydrogenase (SCAD) gene of a patient with SCAD deficiency, suggesting that the conserved arginine is crucial for formation of active enzyme in the straight-chain acyl-CoA dehydrogenases.     

Structural elucidation and properties of 8alpha-(N1-histidyl)riboflavin: the flavin component of thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase.

In addition to 8alpha-(N3-histidyl)riboflavin, 8alpha-(N1-histidyl)riboflavin is also formed during the reaction of Nalpha-blocked histidine with 8alpha-bromotetraacetylriboflavin in a yield of 20-25% of the total histidylflavin fraction. The properties of 8alpha-(N1-histidyl)riboflavin are inditical with those of the histidylflavin isolated from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase but differ from those of 8alpha-(N3-histidyl)riboflavin. These properties include pKa of fluorescence quenching, electrophoretic mobility at pH 5.0, stability to storage, and reduction by NaBH4. Proof for 8alpha substitution is shown by the electron paramagnetic resonance and electron-nuclear double resonance spectra of the cationic semiquinone form, as well as by the proton magnetic resonance spectrum of the oxidized form. The site of histidine substitution by the 8alpha-methylene of the flavin moiety was shown by methylation of the imidazole ring with methyl iodide, cleavage of the methylhistidine-flavin bond by acid hydrolysis at 150 degrees C, and identification of the methylhistidine isomer by electrophoresis. 3-Methylhistidine is the product from the N1-histidylflavin isomer, while 1-methylhistidine is produced from the N3 isomer. The flavin product from reductive Zn cleavage of either isomer has been identified as riboflavin. The compound obtained on acid treatment of 8alpha-(N3-histidyl)riboflavin (previously thought to be the N1 isomer) differs from the parent compound only in the ribityl side chain, since chemical degradation studies show 1-methylhistidine as a product and a flavin product which differs from riboflavin only in mobility in thin-layer chromatography, but not in absorption, fluorescence, and electron paramagnetic resonance spectral properties. Proof that acid modification involves only the ribityl chain has come from the observations that alkaline irradiation of this flavin yields lumiflavin, that the proton magnetic resonance spectrum of the compound differs from that of riboflavin in the region of the ribityl proton resonance, and that its periodate titer is lower than that of authentic riboflavin. The identity of 8alpha-(N1-histidyl)riboflavin with the histidylflavin from thiamine dehydrogenase and beta-cyclopiazonate oxidocyclase shows that both isomeric forms of 8alpha-histidylflavin occur in nature.     

Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase with 3-thiaoctanoyl-CoA

The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.     

Mechanistic studies on the dehydrogenases of methylotrophic bacteria. 2. Kinetic studies on the intramolecular electron transfer in trimethylamine and dimethylamine dehydrogenase

E.p.r. spectroscopy of the trimethylamine and dimethylamine dehydrogenases of Hyphomicrobium X indicates that the substrate-reduced forms of these enzymes exist in the triplet state, which arise through interaction of a reduced [4Fe-4S] cluster and flavosemiquinone, with e.p.r. signals which differ in detail from those of the trimethylamine dehydrogenase of bacterium W3A1. Under certain conditions the intramolecular electron transfer between the flavoquinol form of 6-S-cysteinyl-FMN and the [4Fe-4S] cluster in all three dehydrogenases was much slower than the preceding reduction of the flavin to the flavoquinol form. Trimethylamine dehydrogenases from both organisms show a time-dependent broadening of the e.p.r. signals centred around g = 2 after mixing with trimethylamine. The broadening of the e.p.r. signals could be correlated with an unexpected dependence of the rate of formation of the triplet state on substrate concentration. A model which accounts in a qualitative manner for the substrate dependence of the formation of the triplet state in the trimethylamine dehydrogenase of Hyphomicrobium X is proposed. The binding of the substrate to the reduced form of the enzyme seems to result in a conformational change of the enzyme to a form in which the rate of intramolecular electron transfer is decreased. This finding may be correlated with the observation of hyperbolic substrate inhibition for both trimethylamine dehydrogenases. The results indicate the transfer of an electron to the [4Fe-4S] cluster to be an obligatory step in catalysis and suggest that the transfer of electrons from these enzymes to electron acceptors is mediated solely through the [4Fe-4S] cluster.     

Synthesis and structure-activity relationship studies on a novel series of naphthylidinoylureas as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT)

The synthesis and structure-activity relationships of N-phenyl-N'-[3-(4-phenylnaphthylidinoyl)]urea derivatives 3 as a novel structural class of potent ACAT inhibitors is described. A 3-methoxy group substituted on the naphthylidinone 4-phenyl ring, together with a 1-N-(n)butyl substitution, SM-32504 (3m), gave a potent ACAT inhibitor, in vitro, respectively. The most potent compound, SM-32504 (3m), decreased the serum cholesterol level significantly in a high fat and high cholesterol-fed mouse model.     

Bioluminescence emission of bacterial luciferase with 1-deaza-FMN. Evidence for the noninvolvement of N(1)-protonated flavin species as emitters

The reaction of reduced 1-d-FMN with oxygen and decanal results in bioluminescence with kinetic and spectral properties similar to those of the reaction with FMNH2, even though the spectral (absorbance, fluorescence) and chemical properties of the oxidized forms differ greatly. This emission, which is about 10-15% as efficient as with FMNH2, is postulated to involve the intermediacy of the corresponding 4a-hydroperoxide, the fluorescence of which occurred transiently. The N(1) protonated species had been proposed as the emitter in the reaction with FMNH2, but the 1-deaza analog cannot be protonated at the corresponding position, thus excluding this possibility.     

Acidianus ambivalens type-II NADH dehydrogenase: genetic characterisation and identification of the flavin moiety as FMN

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely (31)P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8alpha-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.     

Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain)

The present study tested the hypothesis that calpain is responsible for the limited proteolytic conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). We compared the effects of various proteases on the activity and molecular weight of a purified preparation of xanthine dehydrogenase from rat liver. In agreement with previous reports, trypsin treatment produced a complete conversion of XD to XO accompanied by a limited proteolysis of XDH from an Mr of 140 kD to an Mr of 90 kD. Treatment with calpain I or calpain II did not produce a conversion from XD to XO nor did it result in partial proteolysis of the enzyme. Similarly, trypsin treatment partially degraded a reversibly oxidized form of xanthine dehydrogenase while calpain I or calpain II were ineffective. The possibility that an endogenous inhibitor prevented the proteolysis of XDH by calpain I or II was excluded by verifying that brain spectrin, a known calpain substrate, was degraded under the same incubation conditions. The results indicate that calpain is not likely to be responsible for the in vivo conversion of XD to XO under pathological conditions.     

Evidence for the direct transfer of the carboxylate of N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) to generate 4-carboxy-5-aminoimidazole ribonucleotide catalyzed by Escherichia coli PurE, an N5-CAIR mutase

Formation of 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) in the purine pathway in most prokaryotes requires ATP, HCO3-, aminoimidazole ribonucleotide (AIR), and the gene products PurK and PurE. PurK catalyzes the conversion of AIR to N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) in a reaction that requires both ATP and HCO3-. PurE catalyzes the unusual rearrangement of N5-CAIR to CAIR. To investigate the mechanism of this rearrangement, [4,7-13C]-N5-CAIR and [7-14C]-N5-CAIR were synthesized and separately incubated with PurE in the presence of ATP, aspartate, and 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetase (PurC). The SAICAR produced was isolated and analyzed by NMR spectroscopy or scintillation counting, respectively. The PurC trapping of CAIR as SAICAR was required because of the reversibility of the PurE reaction. Results from both experiments reveal that the carboxylate group of the carbamate of N5-CAIR is transferred directly to generate CAIR without equilibration with CO2/HCO3- in solution. The mechanistic implications of these results relative to the PurE-only (CO2- and AIR-requiring) AIR carboxylases are discussed.